Genome-wide profiling of in vivo RNA structure at single-nucleotide resolution using structure-seq

Structure-seq is a high-throughput and quantitative method that provides genome-wide information on RNARNARNA structure at single-nucleotide resolution. Structure-seq can be performed both in vivo and in vitro to study RNARNARNA structure-function relationships, RNARNARNA regulation of gene expression and RNARNARNA processing. Structure-seq can be carried out by an experienced molecular biologist with a basic understanding of bioinformatics. Structure-seq begins with chemical RNARNARNA structure probing under single-hit kinetics conditions. Certain chemical modifications, e.g., methylation of the Watson-Crick face of unpaired adenine and cytosine residues by dimethyl sulfate, result in a stop in reverse transcription. Modified RNARNARNA is then subjected to reverse transcription using random hexamer primers, which minimizes 3' end bias; reverse transcription proceeds until it is blocked by a chemically modified residue. Resultant cDNANAs are amplified by adapter-based PCRPCRPCR and subjected to high-throughput sequencing, subsequently allowing retrieval of the structural information on a genome-wide scale. In contrast to classical methods that provide information only on individual transcripts, a single structure-seq experiment provides information on tens of thousands of RNARNARNA structures in similar to 1 month. Although the procedure described here is for Arabidopsis thaliana seedlings in vivo, structure-seq is widely applicable, thereby opening new avenues to explore RNARNARNA structure-function relationships in living organisms.